Parts of this chapter are intended for publication
Data and results in this chapter were gained in cooperation with Prof. Dr. Johannes Kauffold and the Faculty of Veterinary Medicine of the University of Leipzig. Investigations on swine were performed by Catherine Poser and Rosa Stark, PhD students of Prof. Dr. J. Kauffold with the help of Dr. Haukur Lindberg Sigmarsson and Dr. Matthias Hoops. Analysis of the obtained data with corresponding statistics was performed by me at the LMU in Munich.
119
Abstract
For evaluation of an ecologically compatible alternative intended for cycle blockage and estrus control, G [6-D-Phe]-loaded lipid microparticles were tested in vivo. Two different reconstitution media were applied to investigate applicability and syringeablilty of the suspension, revealing suspension properties for a PVP containing medium. In general, lipid-based microparticles were well tolerated throughout the studies with only mild reactions on injection sites. To evaluate the appropriate dose, 750 µg per animal and 3750 µg per animal were administered via different formulations. The low peptide concentration was sufficient for a reliable cycle blockage in swine. It was possible to achieve a cycle blockage between 6.4 ± 0.2 d with D114 + 5 % Span 40 and 14.2 ± 4.9 d with D118 + 10 % GMS. Onset of follicular growth could be controlled between 7 and 17 d depending on the formulation, with significantly delayed onset of follicular growth in the group treated with D114 + 5 % Span 40 microparticles. Ovulation was significantly reduced using the high dose formulations compared to placebo. The incidence of temporary and permanent ovarian cysts was higher in the treatment groups compared to the controls, without statistical evidence. Thus, it could be demonstrated that G [6-D-Phe] loaded lipid microparticles are a promising environmentally sustainable alternative for cycle blockage and estrus control in swine.
120
Introduction
For economic reasons in livestock breeding, animals are estrus synchronized to allow a fixed-time insemination at a predictable fixed-time point [1]–[4]. A variety of gestagens is currently used to achieve a cycle blockage [5]–[7]. Allyl trenbolone, also referred to as altrenogest, was one of the first synthetic progestogens daily administered to influence the estrus in gilts. A highly reliable estrus control can be achieved when fed in doses between 10 and 20 mg/d per animal over 14 to 19 days [8]–[11]. Further investigations indicate, that a treatment with altrenogest positively influences fertility and litter size of treated animals [12].
Besides the use in livestock breeding, gestagens like levonorgestrel and drospirenone are also active ingredients of hormonal contraceptives for women [13] [14]. Concerns about the intensive use of gestagens arise from their entry into the environment harming fish reproduction which results in infertility and masculinization [15]–[17]. Carlsson and co-workers tested a variety of drugs towards their effect on the water environment and identified the gestagen norethisterone as a substance with high toxicity for water organisms with possible long-term effects [18]. The problem is more pronounced due to the absent or if only minimal metabolization [16] [19] [20]. Therefore, there is a need for alternatives in the food-producing animal industry, where large amounts of gestagens are fed daily and which can enter the surrounding runoff, ground and water [19] [21]–[23]
To achieve estrus control with minimal effects on the environment, the application of GnRH analogues, like G [6-D-Phe] is of high interest. The original GnRH decapeptide pGlu1-His2-Trp3-Ser4-Tyr5-Gly6-Leu7-Arg8-Pro9-Gly10-NH2 and G [6-D-Phe] are completely metabolized to non-toxic components [24]–[26]. The introduction of bulky and hydrophobic amino acids in position 6of the peptide backbone is an important structural change to reduce degradation and increase bioactivity [25] [27] [28]. G [6-D-Phe] with D-Phe in position 6 instead of Gly is a so called superagonist for GnRH I, which has a 2.5-fold longer half-life compared to GnRH and shows higher resistance to degradation, due to modification in the cleavage point for endogenous peptidases [26] [27] [29].
A drawback of G [6-D-Phe] against gestagens in estrus synchronization is the need for parenteral application. Since daily injection is not desirable in food animal health, a sustained release system appears mandatory. Different delivery systems for GnRH analogues have been reported in literature for a diversity of mammalian species [30] [31]. Wenzel et al. incorporated GnRH in a sustained release poloxamer gel to achieve a sustained release after depot injection
121 in cattle and to protect the drug against degradation [32]. Further approaches with a successful achievement of synchronized estrus was reported in beagle bitches with the incorporation of leuprolide acetate into PLGA microparticles followed by treatment with fertirelin [33].
Furthermore, a long-term suppression of sexual functions in male and female dogs can be achieved with Suprelorin® (Virbac), a deslorelin containing implant based on hydrogenated palm oil and Phosphatidylcholine. Similarly, the sexual functions in the macaque could be controlled for 3 to 5 months with the delivery of buserelin via an implant [30] [31] [34]–[36].
These approaches and promising results from previously performed in vitro studies on G [6-D-Phe] loaded microparticles pathed the way for in vivo testing of these systems in swine.
Effects on cycle blockage, ovulatory behavior, side effects and biocompatibility were to be evaluated [37]. Based on a previous study on continuous application of G [6-D-Phe] via pumps, consequently 750 µg or 3750 µg per animal were tested [38].
122
Materials and Methods
Materials
Gonadorelin [6-D-Phe] (G [6-D-Phe]) was donated by Veyx Pharma GmbH (Schwarzenborn, Germany). Triglycerides Dynasan 114 (trimyristin, D114), Dynasan 116 (tripalmitin, D116) and Dynasan 118 (tristearin, D118) were kindly provided from Cremer Oleo (Witten, Germany). Glycerol monostearate (GMS) with a monoester content of 40-55 % was purchased from Caelo (Caesar & Loretz, Hilden, Germany). Sorbitan monopalmitate (Span 40) was purchased from Croda (Nettetal, Germany). Mannitol and centrifuge tubes were purchased from VWR International GmbH (Darmstadt, Germany). Distilled water produced with a distillation apparatus (Wagner und Munz, München, Germany) was used for preparation of reconstitution media. Highly purified water was produced using a Milli-Q Water system, (Millipore, MA, USA). The gelling agent Tylopur® C 300 (sodium carboxymethylcellulose, Na CMC) was kindly donated from Clariant (Muttenz, Switzerland) and Kollidon® 90F (Polyvinyl pyrrolidone, PVP) was acquired from BASF (Ludwigshafen, Germany). Polysorbate 20 (PS 20) and polysorbate 80 (PS 80) were purchased from Merck KGaA (Darmstadt, Germany).
Animals
German Landrace x Piétrain gilts with a mean weight of 143.8 kg and an average age of 211 d at the beginning of the study were used. The animals were weaned from the mother after 28 d and kept at a flat deck for further 49 d. Afterwards, the animals were housed in a separate stable in pens à 2 animals and fed dependent on the age according to the guidelines of the Deutsche Landwirtschaftliche Gesellschaft (DLG) [39].
Preparation of G [6-D-Phe] loaded microparticles by spray congealing
Lyophilized G [6-D-Phe] was filled in high grade stainless steel beakers and milled using a Retsch® Cryomill (Retsch Technology, Haan, Germany) with two stainless steel balls (10 mm diameter) applying a precooling time of 10 minutes at 5 Hz followed by the actual milling step of 4 minutes at 20 Hz. The obtained powder was aliquoted in 6 R vials, closed with a rubber stopper and crimped. Lipid compounds were melted on a hot plate at approximately 90 °C (D114, D116) and 110 °C (D118), the aliquot of peptide was added and homogenized using a T-10 basic Ultraturrax (IKA Laboratory Technology, Staufen, Germany) for 2 min. The lipid dispersion was poured into the sample container of the B-290 Mini spray dryer (Büchi, Flawil, Switzerland) with additionally installed spray congealing equipment, which was pre-conditioned for 1.5 h prior to the production step. The lipid melt was atomized using nitrogen at a pressure of 6 bar. A spray flow of 414 l/h was used resulting in a filter pressure of 92 mbar.
123 The aspirator power was set to 100 % throughout all experiments. A dehumidifier Deltatherm® LT (Deltatherm® Hirmer GmbH, Much, Germany) was installed keeping the inlet temperature at 13-15 °C.
Determination of drug content
20 mg microparticles were weighed into centrifuge tubes, 2.0 ml of methylene chloride and 2.0 ml of highly purified water were added and the tubes were placed on a horizontal shaking incubator GFL 3031 (Gesellschaft für Labortechnik, Burgwedel, Germany) at 39 °C and 60 rpm for 12 h. Samples of 1 ml were taken from the water phase and analyzed using RP-HPLC. All extractions were performed in triplicate. RP-HPLC analysis was performed using an Agilent RP-HPLC system (Agilent, Santa Clara, CA, USA), supplied with a 250 x 4.6 mm Luna C-8 column(5µm) (Phenomenex, Aschaffenburg, Germany) and a Security Guard™ KJO-482 pre-column (5 µm) (Phenomenex, Aschaffenburg, Germany). Columns were maintained at 40 °C using the following gradient:
Time [min] A [%] B [%]
0 65 35
1 65 35
21 65 35
22 10 90
26 10 90
35 65 35
Mobile Phase A: 1000 ml highly purified water + 1 ml trifluoroacetic acid Mobile Phase B: 800 g Acetonitrile + 200 g water + 1.2 ml trifluoracetic acid
Aliquoting of the particles under laminar air flow
Based on the drug content (see 7.3.4) microparticles delivering 750 µg or 3750 µg per animal for the treatment of six animals were weighed under laminar air flow (LAF) into sterile 10 R vials, closed with rubber stoppers and crimped.
Preparation of the reconstitution medium
For the pre-clinical studies, two different reconstitution media were used. Firstly, the reconstitution medium consisted of 1 % Na CMC and 0.02 % PS 20. The solution was filtrated through a 5 µm-syringe filter into sterilized 10 R glass vials under LAF closed with sterile rubber stoppers and sterilized in a table autoclave GTA 50 (Medizin und Labortechnik Fritz Gössner, Hamburg, Germany). Optimized reconstitution medium was prepared under LAF
124 conditions using the above-mentioned apparatus by adding 0.5 % PS 80, mannitol for isotonization and 3 % Kollidon® 90 F to distilled water while stirring followed by a sterile filtration through a 0.22 µm syringe filter. Each vial contained 20 ml of reconstitution medium and was closed with sterilized rubber stoppers and crimped. The vials were stored in the refrigerator until application.
Viscosity measurements
Both reconstitution media were analyzed on a rotation viscometer MCR 100 rheometer (Physica, Anton Paar, Ostfildern, Germany) applying the cone-plate-method at 25 °C. Shear rates between 1 s-1 and 100 s-1 were applied. Both pure reconstitution medium as well as particle suspension was tested. 100 mg of particles were suspended in 4.0 ml of reconstitution medium.
Each measurement was performed in triplicate.
General study set-up and pre-treatment of the animals
Both in vivo studies were conducted at the Versuchsgut Oberholz of the University of Leipzig between September 2014 and August 2015. Gilts specifically bred for this purpose at age of 180 days were brought in contact with a boar to detect possible estruses. The study started with the ultrasound examination to assure onset of puberty in all participating animals. If this was not the case, puberty was induced using Intergonan® 240 IE/ml (equine chorionic gonadotropin, eCG, Intervet, Unterschleißheim, Germany). This treatment was only necessary for six animals out of 25 in the first study. These animals were again examined for detection of puberty status 8 d later [39].
Prior to application, all gilts were estrus-synchronized to assure that every animal reached day 12 of the sexual cycle with the 46 day of the study. For cycle synchronization 20 mg/animal Regumate® (altrenogest, Intervet, Unterschleißheim, Germany) were applied starting on day 11 for 28 d. 40 h after the last altrenogest application, 1.000 IE Intergonan® were injected i.m. to induce follicular growth. Ovulation was induced by intramuscular injection of 50 µg/ml Gonavet® Veyx (G [6-D-Phe], Veyx Pharma GmbH, Schwarzenborn, Germany). With day 46 of the study, gilts were randomized into five groups, consisting of four treatment groups and one control group. The injection of the different formulations started on the same day. Table 7-1 gives an overview on formulations tested in the first and second pre-clinical study.
125 Table 7-1: Overview of applied treatments and G [6-D-Phe] concentrations per group. Groups 1-5 were investigated during the first pre-clinical study in September 2014, groups 6-10 were investigated in August 2015 in the second study
Pre-clinical study 1 Triglyceride Additive/concentration G [6-D-Phe]
concentration [µg/animal]
1 D118 - 0 (Placebo)
2 D116 GMS/10 % 750
3 D118 GMS/10 % 750
4 D116 GMS/10 % 3750
5 D118 GMS/10 % 3750
Pre-clinical study 2 Triglyceride Additive/concentration G [6-D-Phe]
concentration [µg/animal]
6 D114 Span 40/5 % 0 (Placebo)
7 D116 Span 40/5 % 750
8 D114 GMS/5 % 750
9 D114 Span 40/5 % 750
10 D114 Span 40/10 % 750
From day 34 on, the gilts were examined clinically and sonographically with focus on cycle state. Moreover, an additional detection of possible estruses was performed with the presence of a boar. Injection of microparticle formulations was performed on day 46 followed by daily visible inspection of injection sites. All animals were investigated at least 25 days and maximum 31 days after injection of the formulations to detect the end of cycle blockage and following follicular growth and ovulation [39].
Reconstitution and application of the suspension
Microparticles and reconstitution medium were delivered in two separate vials and mixed prior to application. In general, 50 to 100 mg microparticles (depending on the actual drug load) were mixed with 2.0 ml of corresponding reconstitution medium. Each vial contained single doses for potentially six animals. The vials were gently shaken for homogenization avoiding foam formation. Previous experiments suggested a manual shaking for 2 min, whereas the practical application needed reconstitution times of 15 min until homogeneity was reached with the high dose formulations. The multi-dose containers were assembled with the injection revolver and the respective amount of suspension was injected into the lateral neck muscles. High dose
126 formulations (3750 µg/animal) were suspended in 4 ml of reconstitution medium and 2 x 2 ml were injected [39].
General examination and evaluation of injection sites
Gilts were investigated concerning interior body temperature with the means of rectal temperature measurement, food intake, general condition and clinical signs of disease between days 34 to 72/78. Injection sites were evaluated with special focus on redness, pain, swelling and temperature. Results were documented photographically [39].
Determination of estrus control
Besides contact with a boar, estrus was detected via redness and swelling of the genitals, as well as beginning and end of “standing” according to a pre-defined schedule. “Standing” was detected via established tests, e.g. back-pressure test by instructed personnel. This data was gained as additional information to assure a gilt´s estrus [39].
Ultrasound examination of ovaries
Ultrasound examinations were performed using a Fazone CB device (Fujifilm, Tokyo, Japan) equipped with a convex transducer type C 9-3, achieving a frequency of 3-9 MHz. For our investigations, a frequency of 6 MHz was used, which assured a penetration depth of 10 cm.
Overall gain was set at 84 dB. For examination, the animals were brought into a custom-made box and immobilized by feeding. Ultrasound was performed transcutaneously according to Kauffold et al., by positioning the transducer from the right ventro-lateral abdominal wall in dorsal direction to the last pair of teats and cranial to the hind leg [40]. Structures like follicles, corpora haemorrhagica and corpora lutea were monitored and documented. The term ovarial cysts was used for structures consisting of growing follicles with a diameter larger than 10 mm showing no ovulation. These structures were persistent besides ovulating follicles (solitary cysts) or exclusively, then called polycystic ovary degeneration (POD). If they were persistent solely over a defined period without influencing the cycle blockage, they were called temporary cysts. If monitored over the whole-time period, they were called permanent cysts. Ovulation was set as complete, if pre-ovulatory follicles vanished and just corpora haemorrhagica were present. Each measurement consisted of at least three representative follicles and if present, three corpora lutea to depict the onset of new follicle growth [41]. This value consisted of the mean value out of three single measurements for each day, respectively [39].
Statistical Analysis
Statistical Analysis was carried out using SigmaPlot (Systat Software Inc., San Jose, CA, USA).
127
Results and Discussion
Characterization of reconstitution behavior
Viscosity measurements were conducted to evaluate applicability of the particle suspensions (Table 7-2).
Table 7-2: Viscosities of reconstitution media and final suspensions
Formulation Viscosity [Pas] Shear rate [s-1]
Na CMC 1 % medium 0.041 (± 0.002) 1 Na CMC 1 % suspension 20.9 (± 13.1) 1 Na CMC 1 % medium 0.036 (± 0.002) 100 Na CMC 1 % suspension 0.050 (± 0.006) 100
PVP 3 % medium 0.014 (± 0.007) 1
PVP 3 % suspension 8.0 (± 4.2) 1
PVP 3 % medium 0.007 (± 0.004) 100
PVP 3 % suspension 0.012 (± 0.002) 100
In literature, shear thinning and dilatancy are reported for suspensions, depending on medium and concentration of the dispersed phase [42] [43]. Both reconstitution media and suspensions showed shear thinning behavior. Shear thinning was more pronounced when Na CMC was used as thickening agent compared to PVP, possibly due to linear orientation of the colloids like cellulose upon mechanical stress [44]. Na CMC based reconstitution medium showed a higher viscosity without particles at low shear rates (0.041 ± 0.002 Pas), as well as after suspension (20.9 ± 13.1 Pas) compared to the PVP based medium (0.014 ± 0.005 Pas without particles, 8.0 ± 4.2 Pas with particles). With suspension of particles, both media showed a remarkably reduced viscosity after exposure to shear stress in comparison to the unsheared sample indicating an orientation of the particles upon shearing. Foam formation was only noticeable upon shaking the Na CMC based formulation with the potential of particle loss at the surface and inhomogeneity (Figure 7-1). Thus, the reconstitution medium based on PVP appeared to be more suitable for reconstitution of the particles, especially with a suspension start viscosity of 8 Pas, and a marked shear thinning to 0.012 Pas, which should enable easy syringeability and injectability.
128 Applicability of the final suspensions in vivo
For both studies two different compositions of reconstitution media were evaluated.
Figure 7-1: Particles suspended in reconstitution medium (Na CMC 1 %, PS 20 0.02 %) with formation of foam due to manual shaking and absorption of particles to the surface (A) kindly provided from the Faculty of Veterinary Medicine, University of Leipzig. Placebo formulation (D114 + 5 % Span 40) during homogenization in reconstitution medium (PVP 3 %, PS 80 0.5 %, mannitol 5 %) (B).
Homogenous suspension of particles in PVP-based reconstitution medium (C)
Na CMC-based medium did not show satisfying reconstitution properties specifically for the high dose formulations (3750 µg/animal). Particles showed a marked tendency towards agglomeration (Figure 7-1 A). To achieve a homogenous suspension, the vials needed to be shaken manually for 15 to 30 min, which is not practicable for a market product. Furthermore, the formation of foam made it difficult to withdraw the desired volume, which necessitated improvement for the second pre-clinical study. Formation of foam could be reduced by exchanging Na CMC with PVP, as well as PS 20 by PS 80 and despite increasing the concentration of emulsifier from 0.02 to 0.5 %. All verum formulations of the second pre-clinical study could be suspended and injected. The placebo formulation still required longer reconstitution times (Figure 7-2).
C
A B C
129 Figure 7-2: Particle suspension in optimized reconstitution medium (PVP 3 %, PS 80 0.5 %, mannitol 5 %) prior to the application in the injection revolver (A). Injection of the suspension into the pig´s lateral neck muscle (B). Marking of the injection site for further examination and documentation of adverse reactions (C)
Biocompatibility and documentation of adverse drug reactions
Figure 7-3: Adverse reactions documentated in first (A) and second pre-clinical study (B). All values are calculated as percentage of the treated animals (control: 5, black bars; treatment 20 animals, grey bars). Statistics were performed using a Mann-Whitney-Rank-Sum-test
The lipid microparticles were well tolerated, which went in accordance with observations from other researchers [45]–[47]. None of the gilts showed pain at the injection site in both control and treated groups. Throughout the first study, both control and treatment groups did not show signs of illness or fever. In the second study one gilt showed fever symptoms directly after injection and another after 24 d. Redness of the injection site could be detected in both studies, without significant difference between control and treatment groups, possibly reasoned by rapid injection without fixation of the animals. In both pre-clinical trials, more animals of the control groups showed redness at the injection sites. A significant difference (P-value: 0.04) was observed concerning warmth of injection sites in the second study, where a higher percentage of the control group showed increased temperature, potentially due to reconstitution problems.
Adverse Reaction
Redness Warmth Swelling Pain
Percentage
0 20 40 60 80 100
Adverse Reactions
Redness Warmth Swelling Pain
Percentage
0 20 40 60 80 100
*
A B C
A B
130 Mild swelling was observed in all animals in both control and treatment groups during the first study, which could be minimized to 60 % due to better reconstitution properties. Two animals showed an intermediate swelling persisting for 4 d (treatment) and for 13 d (control).
Isotonization with 5 % mannitol and replacement of Na CMC with PVP might be reasons for the better compatibility. In general, the treatment with G [6-D-Phe] did not show a significant increase in occurrence of adverse reactions compared to control.
Effects on cycle blockage in vivo using different formulations and G [6-D-Phe]
concentrations
The sustained release G [6-D-Phe]-microparticles were evaluated with respect to minimal effective concentration as well as effect on cycle blockage and estrus control. Both placebo formulations of the first (D118) and second pre-clinical study (D114 + 5 % Span 40) did not influence the cycle (Figure 7-4).
D118 Placebo D116 + 10 % GMS 750 µg
D118 + 10 % GMS 750 µg D116 + 10 % GMS 3750 µg
D118 + 10 % GMS 3750 µg
0 5 10 15 20 25 30 35
Cycle blockage [d]
D114 + 5 % Span 40 Placebo D116 + 5 % Span 40 750 µg
D114 + 5 % GMS 750 µg D114 + 5 % Span 40 750 µg
D114 + 10 % Span 40 750 µg
0 5 10 15 20 25 30 35
Cycle blockage [d]
Figure 7-4: Duration of cycle blockage after injection of lipid based microparticle formulations. Results of the first pre-clinical study using G [6-D-Phe] 750 µg/animal and 3750 µg/animal in D116/D118 formulations with 10 % GMS (A) as well as D114/ D116 and G [6-D-Phe] 750 µg/animal with addition of GMS and Span 40 tested in the second study (B). Data shown are individual results with mean and SEM. Statistical analysis was performed by one-way ANOVA followed by pairwise multiple comparisons (Holm-Sidak-method)
Using the formulation D116 + 10 % GMS 750 µg/animal, a cycle blockage of 10.0 ± 2.6 d could be achieved with one animal not responding to the therapy. A longer cycle blockage of 14.2 ± 4.9 d was observed in animals receiving the longer chain triglyceride D118 + 10 % GMS 750 µg/animal, with one animal not showing a cycle blockage in the observed time. Increasing the therapeutic dose to 3750 µg/animal resulted in a cycle blockage of 9.8 ± 4.3 d with two gilts not responding to the therapy when treated with D116 + 10 % GMS. The delivery of 3750 µg/
animal G [6-D-Phe] formulated in D118 + 10 % GMS led to a cycle blockage of 15.8 ± 4.2 d in all animals. Due to high variation within the groups receiving the drug containing microparticles, no significant difference between placebo and treatment was observed. The
**
*
* *
*
A B
131 treatment with the higher dose of G [6-D-Phe] did not lead to a longer cycle blockage and did only in case of D118 + 10 % GMS result in a higher response within the group (5/5). In this group, one gilt showed a cycle blockage until the end of the observed time (31 d). In the treatment group with 3750 µg/animal (D116 + 10 % GMS) two animals did not show a response to drug application. Formulations tested in the first study evidenced, that the achievement of cycle blockage with the means of lipid based microparticles is generally possible using the low dose concentration, confirming the study with osmotic pumps [38]. Increasing the G [6-D-Phe]
content did not lead to a significant improvement in therapy success or higher synchronicity.
The highest synchronicity could be observed in the group of D116 + 10 % GMS 750 µg/animal.
Furthermore, the delivery of a higher dose seemed to have the consequence of a permanent cycle blockage, as it was only observed in one of the high-dose groups.
For the second pre-clinical study, four different formulations were tested delivering 750 µg/animal of G [6-D-Phe]. The use of D116 + 5 % Span 40 resulted in a cycle blockage of 4.6 ± 1.5 d, with all animals responding to the therapy but a high variability within responses.
The effect of cycle blockage was significantly different compared to placebo (P-value: 0.017).
The group treated with D114 + 5 % GMS showed a cycle blockage of 2.8 ± 1.4 d with one animal not showing a cycle blockage. Formulation consisting of D114 + 10 % Span 40 led to a cycle blockage of 1 day ± 0.5 days with two animals not responding to the therapy. Best results throughout all studies could be achieved with the formulation D114 + 5 % Span 40, where all treated gilts showed a cycle blockage and the highest synchronicity could be observed (5/5).
Hence, a cycle blockage of 6.4 ± 0.2 d was achieved with a significant difference to the control group (P-value < 0.001) and to the group treated with the formulation D114 + 10 % Span 40 (P-value: 0.005). Consequently, duration of cycle blockage was closest to the desired cycle blockage of 15 days using D116 + 10 % GMS 750 µg/animal, whereas highest synchronicity was observed when D114 + 5 % Span 40 was used. These findings confirmed that it was possible to achieve a cycle blockage with the use of a sustained release G [6-D-Phe]
formulation.
Influence on follicular growth
Onset of follicular growth is another parameter indicating success of the therapy. The follicular growth started after 7 to 7.5 d in both control groups, which represented the regular duration time between diminishment of altrenogest and onset of follicular growth. After treatment with D116 + 10 % GMS 750 µg/animal, follicles started to grow after 14.4 ± 2.8 d. Exchanging the triglyceride to D118 + 10 % GMS 750 µg/animal led to an onset of follicular growth after
132 14.8 ± 3.1 d, leading to a permanent cycle blockage in one treated animal. An increase in G [6-D-Phe] dose to 3750 µg/animal resulted in onset of follicular growth after 14.2 ± 4.1 d (D116 + 10 % GMS) and 17 ± 2.0 d (D118 + 10 % GMS), where one animal did not show follicles after the monitored time. Due to considerably high variability within the groups, no significant difference between treatment and control groups could be detected, although follicular growth was delayed.
D118 Placebo D116 + 10 % GMS 750 µg
D118 + 10 % GMS 750 µg* D116 + 10 % GMS 3750 µg
D118 + 10 % GMS 3750 µg*
0 5 10 15 20 25 30
Onset of Follicular Growth [d]
D114 + 5 % Span 40 Placebo D116 + 5 % Span 40 750 µg
D114 + 5 % GMS 750 µg D114 + 5 % Span 40 750 µg
D114 + 10 % Span 40 750 µg
0 5 10 15 20 25 30
Onset of Follicular Growth [d]
Figure 7-5: Onset of follicular growth after treatment with lipid microparticle formulations. (A) Results of the first pre-clinical study using of G [6-D-Phe] 750 µg/animal and 3750 µg/animal) in D116/D118 formulations with 10 % GMS. (B) Results of follow-up study using D114/D116 with addition of GMS and Span 40 and 750 µg/animal G [6-D-Phe]. Data shown are individual results with mean and SEM.
Statistical analysis was performed by Kruskal-Wallis-one-way ANOVA on ranks (A) and by one-way-ANOVA followed by pairwise multiple comparisons (Tukey-test) (B). * one animal with persistent cycle blockage observed
In the second study, treatment with D116 + 5 % Span 40 resulted in an onset of follicular growth after 11.6 ± 1.5 d. After the treatment with D114 + 5 % GMS follicles occurred after 9 ± 1.9 d. Most promising results were achieved with the use of D114 + 5 % Span 40 particles, where an onset after 13.4 ± 0.2 d was monitored. This difference compared to the control group was significant (P-value < 0.05). Increasing the concentration of Span 40 to 10 % shortened the interval between discontinuation and onset to 7.8 ± 0.6 d. Growing follicles were visible in all treated animals in this study. Thus, the formulation D114 + 5 % Span 40 showed in cycle blockage, synchronicity and onset of follicular growth the most promising results.
Influence of treatment on ovulatory behavior
Another important parameter for a successful reproduction is the occurrence of ovulation of growing follicles. Treatment with altrenogest is known to improve ovulation rates and following litter sizes compared to controls [10]. GnRH and its analogues are indicated in ovulation induction in human and veterinary medicine [48]–[50]. Therapy-associated ovulation within all groups was compared in order to investigate, if the treatment affected ovulation
*
A B
133 behavior in gilts. Both control groups, D118 and D114 + 5 % Span 40 resulted in a 100 %-ovulation observation within the ten tested animals. In the group treated with D116 + 10 % GMS 750 µg/animal only 60 % of treated gilts showed an ovulation. This value was reduced to 40 %, when the formulation D118 + 10 % GMS 750 µg/animal was used instead. A complete anovulatory behavior was observed in the treatment group consisting of D116 + 10 % GMS 3750 µg/animal where none of the treated gilts showed ovulatory behavior. Here, a significant difference compared to the placebo group was monitored (P-value < 0.008). Since the other high dose group did also show a decreased ovulation behavior with statistical significance (20 % of the gilts in this group ovulated, P-value 0.043), the application of the high G [6-D-Phe]
dose could be regarded as responsible for this effect.
Figure 7-6: Observed ovulation percentage after treatment with lipid-based microparticles in the first study. Each bar represents the percentage out of 5 animals per group. Statistical analysis was performed using the one-way ANOVA test followed by a Holm-Sidak pairwise comparison.
In the second study, treatment with D116 + 5 % Span 40 and D114 + 5 % GMS resulted in reduced ovulation percentage to 20 %. The formulation with best performance concerning cycle blockage and follicular growth, D114 + 5 % Span 40, led to an ovulation in 40 % of treated animals. The highest percentage regarding ovulation was observed with the use of D114 + 10 % Span 40 (80 %). In the second study, no statistical evidence of reduced ovulation compared to control was given. Nevertheless, a higher incidence of anovulatory behavior was observed upon treatment with sustained release G [6-D-Phe], whereas only the high dose treatment led to significantly reduced occurrence of ovulation. The possible connection between sustained release treatment with a GnRH-analogue and absence of ovulation and development of ovarian cysts needs to be clarified due to a possible disruption in down-regulation mechanism [51] [52].
D118 Placebo
D116 + 10
% GMS 750 µg
D118
+ 10 % GMS 750 µgl
D116 + 10
% GMS 3750 µg
D118 + 10
% GMS 3750 µg
Ovulation percentage
0 20 40 60 80 100
** *
134 Figure 7-7: Observed ovulation percentage after treatment with lipid-based microparticles in the second pre-clinical study. Each bar represents the percentage out of 5 animals per group. Statistical analysis was performed using the Kruskal-Wallis one-way ANOVA on ranks followed by a pairwise multiple comparison (Tukey-test)
Evaluation of the relation between treatment and the occurrence of temporary and permanent cysts
The occurrence of ovarian cysts is a critical point in estrus synchronization, as it has negative influence on performance of ovaries and success of insemination. Although GnRH analogues are used for the treatment of ovarian cysts [53] [54], therapy-induced formation of abnormal follicular structures is reported in literature in both human and veterinary medicine [51] [55]
[56]. Polycystic ovarian degeneration (POD) is characterized by the occurrence of numerous liquid-filled cysts accompanied by an absence of c. lutea [40]. A higher incidence of POD was observed in estrus synchronization when lower doses (16 mg) of altrenogest were compared to higher doses (20 mg) [4]. Consequently, the parameter cyst formation was included in evaluation of treatment with a sustained release G [6-D-Phe] delivery system. In both control groups, none of the gilts showed permanent or temporary cysts (Figure 7-8). In contrast, treatment with D116/D118 + 10 % GMS 750 µg/animal led to the development of permanent cysts in 40 % of treated gilts (Figure 7-8 A black bars). Increasing the dose to 3750 µg/animal resulted in the development of permanent and temporary cysts in 20 % of treated gilts when treated with the formulation D118 + 10 % GMS 3750 µg/animal.
The formulation D118 + 10 % GMS 3750 µg/animal did not lead to the occurrence of cysts at all. During the second pre-clinical study, three treatment groups (D116 + 5 % Span 40, D114 + 5 % GMS, D114 + 5 % Span 40) showed an occurrence of permanent cysts in 40 % of the animals per group. A lower value of 20 % resulted with the formulation D114 + 10 % Span
D 114 +
5 % Span 40 Placebo
D116
+ 5 % Span 40 750 µg
D114
+ 5 % GMS 750 µg D114
+ 5 % Span 40 750 µg
D114
+ 10 % Span 40 750 µg
Ovulation percentage
0 20 40 60 80 100
135 40. Only the treatment with D116 + 5 % Span 40 microparticles resulted in the development of temporary cysts in 20 % of the animals.
Figure 7-8: Percentage of observed cysts (permanent: black bars, temporary: grey bars) during the treatment with G [6-D-Phe]-loaded lipid based microparticles monitored in the first pre-clinical study (A) and the follow-up study (B). Each bar represents the percentage out of the population of 5 animals per group. Statistical analysis was performed using the Kruskal-Wallis one-way ANOVA test on ranks Kauffold et al. reported a very low incidence of the severe POD in gilts compared to sows upon estrus synchronization with gestagens and gonadotropins [40]. Besides the occurrence of single temporary or permanent cysts in our experiments, the severe POD did not occur. Although there was no statistical evidence, treatment with sustained release G [6-D-Phe] and the occurrence of cysts should be evaluated in a larger group of treated animals. The occurrence of follicular cysts is reported to be a severe disorder followed by infertility [40]. In literature, the treatment with LHRH-analogues is indicated in the presence of ovarian cysts in dairy cows also in combination with cloprostenol to treat these abnormal follicles [53] [57]. A possible explanation for this contradictory effect might be, that a treatment with gonadotropins possibly changes LH:FSH ratio, necessary for normal follicles as stated by Breen and Knox [58]. Also in human medicine, an increased development of follicular and luteal cysts was reported as complication during preparation for in vitro fertilization by the application of GnRH-analogues [55] [56] [59].
Conclusion
Our results showed that it was possible to achieve a cycle blockage with a sustained release formulation of G [6-D-Phe] in swine. Spray congealed solid lipid microparticles are a suitable
D118 Placebo
D116
+ 10 % GMS 750 µg D118
+ 10 % GMS 750 µg D116
+ 10 % GMS 3750 µg
D118
+ 10 % GMS 3750 µg
Percentage
0 10 20 30 40 50
D 114 + 5 % Span 40 Placebo
D116 + 5 % Span
40 750 µg D114
+ 5 % GMS 750 µg
D114 + 5 % Span
40 750 µg
D114
+ 10 % Span 40 750 µg
Percentage
0 10 20 30 40 50
B A
136 delivery system to influence and control the estrus. Particles were prepared without using organic solvents and were evaluated to be well tolerated and highly biocompatible in both in vivo studies.
Reconstitution and application of the microparticular suspension was difficult with the use of a Na CMC-based medium. Especially when high particle doses (3750 µg/animal) needed to be administered, foam formation and attachment of particles to the liquid/air interface occurred.
These drawbacks could be avoided using the optimized, PVP-based medium with higher emulsifier concentration, facilitating reconstitution and application.
The first study confirmed the successful estrus control with the use of 750 µg/animal G [6-D-Phe] resulting in a mean cycle blockage of 10 to 16 d with an onset of follicular growth after 15 to 17 d. These values came very close to the desired cycle blockage of 15 d. Here, a treatment with D116 in combination with 10 % GMS resulted in the highest synchronicity within the group. Compared to the placebo treatment, a significant reduction in ovulation was observed after the application of the higher dose (3750 µg/animal) and without statistical evidence after treatment with 750 µg/animal. Three out of four treated groups showed development of temporary and permanent follicular cysts without statistic relevance compared to controls.
Having proven our concept with the first pre-clinical study, the second study focused on the application of 750 µg/animal via four different formulations to narrow the time window to the desired 15 d with a higher synchronicity. Best results were achieved with the formulation D114 + 5 % Span 40, where a duration of 6 d and the highest synchronicity in all trials could be observed. Follicular growth was setting in between 9 and 13 d after application. No significant differences in ovulation behavior between treatment and control was observed. All treatment groups showed development of ovarian cysts indicating a correlation between treatment with G [6-D-Phe] and the occurrence of these structures, without statistical evidence.
In summary, it is possible to achieve a cycle blockage in variable time frames with the application of G [6-D-Phe]-loaded microparticles by variating the type of emulsifier, as well as its concentration. Furthermore, we confirmed, which is economically favorable, the successful control of estrus cycle with the low dose G [6-D-Phe].
137
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